The invention relates to the field of modified surfaces, more particularly to silicon surfaces modified with functionalised molecules.
The covalent attachment of organic monolayers to semiconductor surfaces provides a route to passivation and a method to incorporate chemical and biochemical function into solid-state devices to produce bio-sensors or bio-sensor arrays.
Particularly sought after are methods for immobilising bio-molecules with specific binding functions, such as proteins or DNA, on silicon surfaces.
Several methods have been proposed for the preparation of organic layers on silicon surfaces, attached through Sixe2x80x94C and Sixe2x80x94Oxe2x80x94C bonds. The relative susceptibility to hydrolysis of the Sixe2x80x94Oxe2x80x94C bond is a disadvantage of this type of linkage for applications in which an aqueous environment is encountered.
Hydrogen terminated silicon is interesting as a substrate for immobilisation of organic molecules. Chidsey et al. demonstrated that the Si(111)H surface formed by etching with fluoride may be hydrosilylated by immersion in a neat alkene followed by irradiation1. Similar work is disclosed in U.S. Pat. No. 5,429,708 (to Linford et al., issued Jul. 4, 1995). The layers formed are extremely durable.
Bateman et al. have reacted hydrogen-terminated porous silicon surfaces thermally with 1-octene, 1-octyne, 1-undecene and vinyl ferrocene, by refluxing in toluene at 110-180xc2x0 C. The surface-attached ferrocene moiety may be observed by cyclic voltammetry2. Boukherroub et al. use a modification of the method of Buriak et al. to achieve the modification of porous silicon by etching the silicon surface with ammonium fluoride to form a hydrogen-terminated Si(111) surface, followed by hydrosilylation with an alkene in the presence of a Lewis acid catalyst (AlEtCl2)3,4.
Sieval et al. report the thermal reaction of alkenes terminated with ester groups, with an Si(100) hydrogen terminated surface5. The ester groups can be hydrolysed to release a carboxylic acid-modified surface, or reduced with LiAlH4 to produce an alcohol-modified surface. Re-esterification of either of these surfaces by refluxing with an alcohol or carboxylic acid, respectively, in the presence of acid catalyst is possible. The required conditions are unfortunately too harsh to be compatible with most biological molecules.
Hydrogen-terminated Si(100) and Si(111) modified through Sixe2x80x94O linkages have been reported by Zhu et al. A silicon substrate is etched with NH4F, to produced a hydrogen-terminated surface. Exposure of the surface to gaseous chlorine results in a Cl-capped surface, which is subsequently reacted with dodecanol or octadecanol to produce C12 and C18 chains, respectively, immobilised on the Si surface by Sixe2x80x94O bonds6. Zhu et al. has reported a similar procedure, wherein a Cl-capped Si(100) surface is exposed to amine vapour, to produce amines attached to the silicon surface via two Nxe2x80x94Si bonds7. They have reported the use of an analogous procedure to attach molecules to a clean Si(100) surface and a porous silicon surface via Sixe2x80x94N bonds8.
Lewis et al. formed Si(111)xe2x80x94C linkages using a two-step method, whereby an Si(111) surface is first chlorinated, and then reacted with an alkyl Grignard or alkyl lithium reagent9. Boukherroub et al. have demonstrated that similar modification can be achieved by direct reaction of alkyl magnesium bromides with an Si(111) surface10.
The photochemical hydrosilylation of aldehydes with an Si(111)xe2x80x94H surface, to form an Si(111)xe2x80x94OCH2R surface has been reported by Effenberger et al11.
Sailor et al. disclose modification of porous silicon with BSA or protein A via a complicated 8-step method. The linkage to the silicon surface is through an Sixe2x80x94Oxe2x80x94Si linker on the oxidised (i.e. SiO2) surface. Binding of IgG to surface bound protein A is measured by observing the shift in effective optical thickness using interferometry12.
Strother et al. report the modification of Si(111) with non-covalently attached DNA. A hydrogen-terminated Si(111) surface is reacted with an xcfx89-undecylenic acid methyl or trifluoroethyl ester by UV irradiation of a thin film of the ester applied to the surface. The ester functionality is hydrolysed by treatment with potassium t-butoxide in DMSO, to yield a carboxylic acid-modified surface. Polylysine is added, becoming immobilised on the surface through ionic interactions between the polylysine amino groups and the surface carboxylate groups. Thiol-modified DNA is then linked to the polylysine via the bi-functional linker sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate13.
There remains to be found a simple, versatile method for durable covalent attachment of organic molecules to hydrogen-terminated silicon surfaces, under conditions that are compatible with bio-molecules.
It is an object of the invention to provide a method for immobilising molecules on a hydrogen-terminated silicon surface.
It is another object of the invention to provide a silicon surface with an immobilised layer which layer is suitable to be further reacted to attach a bio-molecule.
It is a further object of the invention to provide a silicon surface modified with a bio-molecule.
In a first aspect the invention provides a method for immobilising a desired molecule on a silicon substrate, the method comprising the steps:
(A) providing an Sixe2x80x94H surface on the silicon substrate; and
(B) attaching the desired molecule to the Sixe2x80x94H surface via a covalent bond.
In a second aspect, the invention provides a method for providing a coupling group on a silicon substrate, the method comprising the steps:
(A) providing an Sixe2x80x94H surface on the silicon substrate;
(B) reacting the Sixe2x80x94H surface with a linker-molecule possessing at least one anchor functionality capable of reacting with the Sixe2x80x94H surface to form an Sixe2x80x94C or Sixe2x80x94O linkage, and further possessing at least one coupling group and/or protected coupling group which does not react with the Sixe2x80x94H surface; and
(C) removing unreacted linker-molecule.
In a third aspect, the invention provides a method for immobilising a desired molecule on a silicon substrate, the method comprising the steps:
(A) providing an Sixe2x80x94H surface on the silicon substrate;
(B) reacting the Sixe2x80x94H surface with a linker-molecule possessing at least one anchor functionality capable of reacting with the Sixe2x80x94H surface to form an Sixe2x80x94C or Sixe2x80x94O linkage, and further possessing at least one coupling group and/or protected coupling group which does not react with the Sixe2x80x94H surface;
(C) removing unreacted linker-molecule;
(D) if a protected coupling group is present, deprotecting the protected coupling group; and
(E) reacting the coupling group with the desired molecule.
In a fourth aspect, the invention provides a silicon substrate bearing immobilised coupling groups attached to the silicon substrate by Sixe2x80x94C bonds or Sixe2x80x94O bonds.
In a fifth aspect, the invention provides a silicon substrate bearing a bio-molecule attached to the silicon substrate covalently via Sixe2x80x94C bonds or Sixe2x80x94O bonds.